In radiation therapy, the significant technological disparity between methods used for human treatment and those for laboratory animal irradiation brings into question the suitability of current preclinical radiation research. In an effort to bridge the gap, we have previously employed the Bioengineering Research Partnership (BRP) mechanism and constructed an advanced small animal radiation research platform (SARRP). The SARRP mimics a human treatment machine. It is equipped with on-board x-ray cone-beam CT (CBCT) to guide focal irradiation and is capable of delivering focal radiation to a target with accuracy of 0.2mm. In use for over 2 years, the system has enabled previously infeasible radiation studies with mice. However, it has also become evident that CBCT imaging is poor for localizing small soft tissue targets for irradiation. The hurdle is particularly significant for the increasingly important class of small, pre-palpable, orthotopic, or spontaneous, tumor models growing in a normal tissue environment. We identify that a most powerful solution is to incorporate molecular-optical (bioluminescence and fluorescence) imaging on board the SARRP to complement x-ray CT guidance. Based on a novel rotating single camera design which incorporates CT priors, our specific aims for the 3-year research period are to: (1) Design and construct an integrated x-ray /bioluminescence tomography (BLT) system (with BL as our initial focus) that can function as a standalone research apparatus and also on-board the SARRP to guide focal irradiation; (2) Validate at three academic partner institutions the accuracy and minimum margin expansion with which on-board x-ray/BL tomography can be used to localize and ensure coverage of a soft tissue target. We submit our proposal in response to PAR-10-169 for academic-industrial partnership for in vivo imaging research. A multi-disciplinary team is formed with expertise in radiation sciences, robotics, optical imaging and manufacturing. Our major industrial partner is Gulmay Medical Ltd. which has an existing partnership with Hopkins in commercializing the current SARRP product. In addition to Hopkins' system, two commercial grade prototypes will be constructed by Gulmay. In fulfillment of the innovation requirements of the PAR-10-169 program, the University of Pennsylvania and University of Virginia participate as minor partners and represent the end-users to conduct in-field validation of these two systems. In addition to Gulmay, Caliper Life Sciences also participates as industrial consultant to capture commercialization opportunities for dissemination.